Processes for preparing oxathiazin-like compounds

ABSTRACT

Oxathiazin-like compounds, processes for making new oxathiazin-like compounds, compounds useful for making oxathiazin-like compounds, and their uses are disclosed. Processes of treating patients suffering from cancers, bacterial infections, fungal infections and/or viral infections by administering oxathiazin-like compounds are also disclosed. These compounds were found to have significantly longer half-life compared to taurolidine and taurultam.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. applicationSer. No. 16/538,344, filed on Aug. 12, 2019, which is a division of U.S.application Ser. No. 15/535,266, filed on Jun. 12, 2017, and granted asU.S. Pat. No. 10,392,355, which is a National Stage Entry ofPCT/IBS2015/059741, filed on Dec. 17, 2015, which claims prioritybenefit of U.S. provisional Appl. No. 62/094,580, filed on Dec. 19,2014, the disclosures of which are incorporated herein by reference intheir entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to new compounds, processes for preparingnew compounds and uses thereof.

Description of the Background Art

Oxathiazin-like compounds are known from U.S. Pat. Nos. 3,202,657 and3,394,109.

There remains a need in the art for new compounds and processes formaking such compounds to provide compounds with more potentantineoplastic and antimicrobial activity, less toxicity and sideeffects, and less resistance to treatment by tumor or microbial cells.

Cutaneous squamous cell carcinoma (cSCC) represents a major problem indermato-oncology because it can be a life-threatening disease andeffective and safe treatments are very limited. There remains asignificant unmet need for suitable therapeutic options for treatingsquamous cell carcinomas.

Tumor-related morbidity and mortality are often due to migration ormetastasis of the original tumor cells to a site away from the originalprimary tumor. Inhibition of tumor and cancer cell migration is along-felt and unmet need in improving the prognosis of patients withtumors. There remains a significant unmet need for suitable therapeuticoptions for preventing, inhibiting and reducing tumor and cancer cellmigration and metastases.

SUMMARY OF THE INVENTION

In accordance with the present invention, new oxathiazin-like compounds,processes for making new oxathiazin-like compounds, compounds useful formaking oxathiazin-like compounds, and their uses are disclosed.

In one aspect, the present disclosure provides a method of reducing orinhibiting cancer cell migration in a subject in need thereof comprisingadministering an effective amount of a compound of the presentdisclosure to the subject. In some aspects, the compound may be acompound of formula I:

wherein R is H, an alkyl, or benzyl.

In one aspect, the compound is

In one aspect, the subject has a tumor, cancerous cells, pre-cancerouscells, or cancer stem cells, is suspected of having a tumor, cancerouscells, pre-cancerous cells, or cancer stem cells, or is at risk ofdeveloping a tumor, cancerous cells, pre-cancerous cells, cancer stemcells or metastases thereof.

In one aspect, the present disclosure provides a method of treating asubject having a squamous cell carcinoma comprising administering aneffective amount of a compound of the present disclosure to the subject.In some aspects, the compound may be a compound of formula I:

wherein R is H, an alkyl, or benzyl.

In one aspect, the squamous cell carcinoma is a cutaneous squamous cellcarcinoma. In one aspect, the compound is administered orally,intravenously, topically, or a combination thereof. In one aspect, thecompound is

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically shows anti-neoplastic activity of one embodiment ofthe invention in a cytotoxicity assay in LN-229 cells.

FIG. 2 graphically shows anti-neoplastic activity of one embodiment ofthe invention in a cytotoxicity assay in SW480 (human colonadenocarcinoma) cells.

FIG. 3A-3C Cytotoxicity induced in murine SMA 560 bulk glioma cellsafter treatment with taurolidine and taurultam (TT). Cytotoxicity wasassessed after 24 h (FIG. 3A) and 48 h (FIG. 3B) of treatment. The EC₅₀values for taurolidine (34.6 μg/ml) and taurultam (19.3 μg/ml) are givenin the lower panel (FIG. 3C). Data are presented as mean values ±SD ofthree independent experiments.

FIG. 4 Cytotoxicity induced by taurolidine and taurultam (TT) in murineSMA560 glioma cancer stem cells (CSC). Data are presented as mean values±SD.

FIG. 5A-5C Cytotoxicity induced in cancer stem cells isolated from fourglioblastoma multiforme (GBM) patients (GBM #3, #4, #5 and #6) aftertreatment for 24 h with taurolidine (FIG. 5A), taurultam (TT) (FIG. 5B)or temozolamide (FIG. 5C). Data are presented as mean values ±SD.

FIG. 6 FTIR spectrum of compound 2244 made according to the presentinvention.

FIG. 7 FTIR spectrum of compound 2250 made according to the presentinvention.

FIG. 8 shows the results of a spheroid toxicity assay for multicellularpancreatic tumor (Panc Tul or BxPC-3) spheroids in which control,taurolidine-treated (500 μM) or compound 2250-treated (1000 μM) sampleswere treated for 48 hours (columns labeled A) and strained to testresidual aggregates (columns labeled B) for stability.

FIGS. 9A and 9B show the results of FACS analysis of the Panc Tulmulticellular spheroid cultures CD133 content.

FIG. 10A shows MiaPaca2 tumor volume upon treatment with control ortaurolidine. FIG. 10B shows MiaPaca2 tumor volume upon treatment withcontrol or compound 2250. FIG. 10C shows PancTu I tumor volume upontreatment with control or taurolidine. FIG. 10D shows PancTu I tumorvolume upon treatment with control or compound 2250.

FIG. 11A is a xenograft model of pancreatic primary tumors (Bo 73)observed for 15 days when treated with control, taurolidine or compound2250. FIG. 11B is a xenograft model of pancreatic primary tumors (Bo 70)observed for 23 days when treated with control, taurolidine or compound2250.

FIGS. 12A and 12B show the effects of GP-2250 in different cSCC celllines, measured by MTT assay. FIG. 12A shows the effects when SCC13cells were incubated with GP-2250 (100, 150, 200, 300 and 400 μmol/l)for 24 h. FIG. 12B shows the effects when A431 cells were incubated withGP-2250 (50, 100, 125, 150 and 200 μmol/l) for 24 h. Values areexpressed as mean value ±SD of 8 independent experiments. Asterisksymbols indicate differences between controls. The significance levelsare graphically shown as follows: ***p≤0.001, **p≤0.01, *p≤0.05, nsp≥0.05 (one-way ANOVA followed by Tukey's post-hoc test).

FIGS. 13A and 13B show the effects of GP-2250 in different cSCC celllines, measured by BrdU assay. FIG. 13A shows the effects when SCC13cells were incubated with GP-2250 (100, 150, 200, 300 and 400 μmol/l)for 6 h. FIG. 13B shows the effects when A431 cells were incubated withGP-2250 (50, 100, 125, 150 and 200 μmol/l) for 6 h. Values are expressedas mean value ±SD of 3 independent experiments. Asterisk symbolsindicate differences between controls. The significance levels aregraphically shown as follows: ***p≤0.001, **p≤0.01, *p≤0.05, ns p≥0.05(one-way ANOVA followed by Tukey's post-hoc test).

FIGS. 14A-14F show the effects of GP-2250 in different cSCC cell lines,measured by FCM analysis. SCC13 cells were incubated with GP-2250 (200,300 and 400 μmol/l) and A431 cells were incubated with GP-2250 (100, 150and 200 μmol/l), both for 24 h. The percentages of viable (FIG. 14A),apoptotic (FIG. 14B) and necrotic SCC13 cells (FIG. 14C) as well asviable (FIG. 14D), apoptotic (FIG. 14E) and necrotic A431 cells (FIG.14F) were determined by FCM-analysis with Annexin V-FITC and Propidiumiodide. Values are expressed as mean value ±SD of 6 independentexperiments. Asterisk symbols indicate differences between controls. Thesignificance levels are graphically shown as follows: ***p≤0.001,**p≤0.01, *p≤0.05, ns p≥0.05 (one-way ANOVA followed by Tukey's post-hoctest).

FIGS. 15A-15D show reduction in cutaneous squamous carcinoma cellmotility in the cell migration assay. FIG. 15A shows representativeimages of the SCC13 cells treated with GP-2250 or negative control at 0h, 6 h, 12 h and 24 h after the scratches were made (×10 magnification)and FIG. 15B shows the related percentage of total gap closure at 0 h,12 h and 24 h. FIG. 15C shows representative images of the A431 cellstransfected with GP-2250 or negative control at 0 h, 6 h, 12 h and 24 hafter the scratches were made at the same starting point (×10magnification) and FIG. 15D shows the related percentage of total gapclosure at 0 h, 12 h and 24 h.

DETAILED DESCRIPTION OF THE INVENTION

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilizing (i.e. not worsening) the state ofdisease, delaying or slowing of disease progression, amelioration orpalliation of the disease state, diminishment of the reoccurrence ofdisease, and remission (whether partial or total), whether detectable orundetectable. “Treating” and “treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment. Inaddition to being useful as methods of treatment, the methods describedherein may be useful for the prevention or prophylaxis of disease.

Concentrations, amounts, and other numerical data may be expressed orpresented herein in a range format. It is to be understood that such arange format is used merely for convenience and brevity and thus shouldbe interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited. Asan illustration, a numerical range of “about 0.01 to 2.0” should beinterpreted to include not only the explicitly recited values of about0.01 to about 2.0, but also include individual values and sub-rangeswithin the indicated range. Thus, included in this numerical range areindividual values such as 0.5, 0.7, and 1.5, and sub-ranges such as from0.5 to 1.7, 0.7 to 1.5, and from 1.0 to 1.5, etc. Furthermore, such aninterpretation should apply regardless of the breadth of the range orthe characteristics being described. Additionally, it is noted that allpercentages are in weight, unless specified otherwise.

In understanding the scope of the present disclosure, the terms“including” or “comprising” and their derivatives, as used herein, areintended to be open ended terms that specify the presence of the statedfeatures, elements, components, groups, integers, and/or steps, but donot exclude the presence of other unstated features, elements,components, groups, integers and/or steps. The foregoing also applies towords having similar meanings such as the terms “including”, “having”and their derivatives. The term “consisting” and its derivatives, asused herein, are intended to be closed terms that specify the presenceof the stated features, elements, components, groups, integers, and/orsteps, but exclude the presence of other unstated features, elements,components, groups, integers and/or steps. The term “consistingessentially of,” as used herein, is intended to specify the presence ofthe stated features, elements, components, groups, integers, and/orsteps as well as those that do not materially affect the basic and novelcharacteristic(s) of features, elements, components, groups, integers,and/or steps. It is understood that reference to any one of thesetransition terms (i.e. “comprising,” “consisting,” or “consistingessentially”) provides direct support for replacement to any of theother transition term not specifically used. For example, amending aterm from “comprising” to “consisting essentially of” would find directsupport due to this definition.

As used herein, the term “about” is used to provide flexibility to anumerical range endpoint by providing that a given value may be “alittle above” or “a little below” the endpoint. The degree offlexibility of this term can be dictated by the particular variable andwould be within the knowledge of those skilled in the art to determinebased on experience and the associated description herein. For example,in one aspect, the degree of flexibility can be within about ±10% of thenumerical value. In another aspect, the degree of flexibility can bewithin about ±5% of the numerical value. In a further aspect, the degreeof flexibility can be within about ±2%, ±1%, or ±0.05%, of the numericalvalue. Numerical quantities given are approximate, meaning that the term“around,” “about” or “approximately” can be inferred if not expresslystated.

As used herein, the term “pharmaceutically acceptable” refers tosolvents, co-solvents, surfactants, carriers, diluents, excipients,buffers, salts, and/or other components that are compatible with theother ingredients of the formulation and are not deleterious to therecipient thereof.

According to certain embodiments, the present invention relates tooxathiazin-like compounds, as well as derivatives thereof and processesand compounds for preparing oxathiazin-like compounds and derivativesthereof.

Oxathiazin-like compounds and derivatives thereof according to certainembodiments of the present invention have antineoplastic activities,antimicrobial activities and/or other activities.

Processes for making oxathiazin-like compounds and derivatives thereofaccording to certain embodiments of this invention provide advantageousmethods for making compounds having antineoplastic activities,antimicrobial activities and/or other activities. In certainembodiments, oxathiazin-like compounds and derivatives thereof areuseful, inter alia, in the treatment of cancers and tumors in a subject,such as a human patient. Accordingly, in certain embodiments the presentinvention also relates to treatment of cancers and tumors usingcompounds described herein. Cancers such as central nervous systemcancers including glioblastoma, glioma, neuroblastoma, astrocytoma, andcarcinomatous meningitis, colon cancer, rectal cancer and colo-rectalcancer, ovarian cancer, breast cancer, prostate cancer, lung cancer,mesothelioma, melanoma, renal cancer, liver cancer, pancreatic cancer,gastric cancer, esophageal cancer, urinary bladder cancer, cervicalcancer, cardiac cancer, gall bladder cancer, skin cancer, bone cancer,cancers of the head and neck, leukemia, lymphoma, lymphosarcoma,adenocarcinoma, fibrosarcoma, and metastases thereof, for example, arediseases contemplated for treatment according to certain embodiments ofthe invention. Drug resistant tumors, for example a multiple drugresistant (MDR) tumor, also are useful in certain embodiments using theinventive compounds, including drug resistant tumors which are solidtumors, non-solid tumors and lymphomas. It is presently believed thatany neoplastic cell can be treated using the methods described herein.

Tumor stem cells (also referred to as cancer stem cells (CSCs)) areconsidered to be the main drivers for the formation of metastases andthe regrowth of tumors after resection.

In certain embodiments, compounds of the present invention are useful,inter alia, in the treatment of tumor stem cells in a subject.

In certain embodiments, compounds of the present invention are useful,inter alia, in the treatment of glioblastoma tumor stem cells in asubject.

In certain embodiments, the invention kills tumor cells and/or CSCs, orinhibits their growth, by oxidative stress, apoptosis and/or inhibitinggrowth of new blood vessels at the tumor site (anti-angiogenesis andanti-tubulogenesis). A primary mechanism of action for killing tumorcells and/or CSCs is oxidative stress. Tumor cells and/or CSCs may alsobe killed by apoptosis according to the invention. At lower bloodconcentrations, compounds according to the invention are effective atinhibiting tumor cell growth by their anti-angiogenic action and theiranti-tubulogenic action, and these compounds are thus useful forpalliative treatment.

Oxathiazin-like compounds and derivatives thereof of the inventionmetabolize much slower in the bloodstream than taurolidine andtaurultam. Accordingly, lower doses of such compounds can beadministered to a patient to achieve similar effects.

It was unexpectedly found that within minutes of exposure totaurolidine, tumor cells react by initiating the program of apoptoticcell death as follows:

-   -   1. The primary insult of Taurolidine to the tumor cell is an        increase of reactive oxygen species (ROS), which is measured        fluorimetrically.    -   2. The induction of oxidative stress by Taurolidine as the        primary step is supported by the finding that the antineoplastic        action of Taurolidine can be prevented by the addition of a        reducing agent such as glutathione or N-acetylcysteine.    -   3. The damage caused by the elevated ROS to the mitochondria of        the tumor cell results in the loss of their membrane potential        and the release of Apoptosis Inducing Factor (AIF).    -   4. AIF is translocated to the nucleus and initiates the        expression of pro-apoptotic genes, which results in the blebbing        of the plasma membrane, in chromatin condensation and DNA        fragmentation, the hallmarks of apoptosis.    -   5. In contrast to normal cells, tumor cells are very sensitive        to oxidative stress. This explains the action of Taurolidine        against a broad range of tumor cells, sparing normal cells.

Compounds of the present invention also are useful, in certainembodiments, in treatment of microbial infections in a subject, such asa human patient. Microbial infections which may be treated accordingcertain embodiments include bacterial infections, fungal infectionsand/or viral infections.

Cancer patients tend to be immunocompromised, making them particularlysusceptible to microbial infections, especially during and/or aftersurgery.

In certain embodiments, compounds of the invention are utilized to treatglioblastoma in a subject.

In certain embodiments, compounds of the invention are utilized to treatS. aureus infection in a subject.

In certain embodiments, compounds of the invention are utilizedaccording to the invention to treat MRSA in a subject.

In certain embodiments, compounds of the invention are utilizedaccording to the invention to treat E. coli in a subject.

In certain embodiments, compounds of the invention are utilizedaccording to the invention to treat H. pylori in a subject, and/orcancer(s) associated with H. pylori in a subject.

In certain embodiments, compounds of the invention are utilizedaccording to the invention to treat HIV in a subject.

In certain embodiments, compounds according to formula I are utilizedaccording to the invention wherein R is H, alkyl, or the like, such asmethyl, ethyl, propyl, (e.g., isopropyl), benzyl or the like.

In certain embodiments, new compound 2250(Tetrahydro1,4,5-oxathiazin-4-dioxide or 1,4,5-oxathiazan-4-dioxide) isprepared and/or utilized according to the invention. An FTIR spectrumfor compound 2250 made according to the present invention is shown inFIG. 8.

In certain embodiments, new compound 2245 is prepared and/or utilizedaccording to the invention.

Compound 2250 prevents and treats stomach tumors, including tumorscaused by or associated with H. pylori, or tumors as a consequence ofmetastasis to the stomach.

The amount of the compounds needed depends on tumor size. In oneembodiment, the invention includes surgically reducing tumor size andtreating with one or more of the compounds. The compound may beadministered before, during or after surgery to reduce tumors. Compoundsaccording to the invention can be administered by any suitable method,including without limitation, by gels, capsules, tablets, IV, IP and/ordirectly to the tumor.

Gels can contain for example 2-4% (e.g., 3%) active compounds of theinvention, such as compound 2250, alone or in combination withtaurolidine/taurultam which also can be administered and present alone,and can be for topical administration. Such gels can be used to treattumors of the skin and mouth, including squamous cell tumors of themouth and skin. Such gels also can be used to treat cervical cancer orcervical dysplasia by being administered in a suppository to the vagina,or by syringe. The invention may include the combination of asuppository carrying an active compound.

Cutaneous squamous cell carcinoma (cSCC) poses a notable threat with itsability to form cutaneous in-transit metastases (>2 cm away from primarytumor) and regional lymph node metastases, followed by distantmetastases, associated with a very poor prognosis and medianpathology-related mortality rate of >70%. An appropriate systemictreatment for metastatic cSCC is still controversial and remainsunclear. Most commonly used therapies in this setting are unspecificcytotoxic chemotherapies mainly using cisplatin and/or 5-fluorouraciland radiotherapy (alone or in combination), even though long-termcurative chemotherapeutic data are still lacking. These therapies areassociated with numerous serious side effects and adverse effectsincluding but not limited to nausea, vomiting, fever, myelosuppression,severe involvement of tissues including neurotoxicity, hepatotoxicity,nephrotoxicity, and ototoxicity. These side effects can form acontraindication for chemotherapy treatments, considering that theconcerned target population is diagnosed with metastatic SCC at a meanage of 70 years and often suffers from significant comorbidities.Immunotherapies such as PD-1 blockade induces a response inapproximately half the patients, but is associated with adverse events(diarrhea, fatigue, nausea, constipation) that usually occur with immunecheckpoint inhibitors.

The present disclosure provides compounds and methods for inhibitingcSCC cell growth and proliferation. The present disclosure providescompounds and methods for avoiding one or more serious side effects andadverse effects disclosed herein. As exemplified in Example 11,compounds of the present disclosure may provide a dose-dependent effectin treating cSCC. For example, increasing concentrations of compoundGP-2250 led to significant and subsequently increasing antineoplasticeffects compared to the untreated control groups in all carried outassays after 24 hours: the maximal achieved viability reduction in A431cells was 95.80% (±0.47) (FIG. 12B) and in SCC13 cells 91.18% (±0.99)(FIG. 12A).

Compounds of the present disclosure are useful in methods of inhibitingcancer cell proliferation, reducing cancer cell motility reduction, andinducing apoptotic and necrotic cell death. For example, as shown in thedata provided herewith, GP-2250 inhibited proliferation, reduced cellmotility reduction, and induced apoptotic and necrotic cell death. Sameproportional dose-effect pattern of substance GP-2250 was detected after24 hours in anti-proliferative effects of BrdU analysis (FIGS. 13A-13B),FCM analysis (FIGS. 14A-14F) and observed in anti-migratory effects incell migration assays, during 24 hours with intermittent measurements(FIGS. 15A-15D). All the highest tested concentrations of GP-2250 (400μmol/l for SCC13 and 200 μmol/l for A431 cells) lead to pronouncedantineoplastic, anti-proliferative and apoptotic effects in all assays,significantly greater than lower administrated concentrations andcompared to untreated controls. In some aspects, the present disclosureincludes administering the compounds of the present disclosure incombination with Cemiplimab-rwlc, Pembrolizumab, Cemiplimab-rwlc,fluorouracil, imiquimod, vismodegib, sonidegib, avelumab, or otherimmunotherapies or chemotherapies. In some aspects, the presentdisclosure includes reducing side effects of administration of one ormore of Cemiplimab-rwlc, Pembrolizumab, Cemiplimab-rwlc, fluorouracil,imiquimod, vismodegib, sonidegib, and avelumab therapy by administeringthe compound of the present disclosure in combination with a reduceddosage of one or more of Cemiplimab-rwlc, Pembrolizumab,Cemiplimab-rwlc, fluorouracil, imiquimod, vismodegib, sonidegib, andavelumab therapy.

Surprisingly, minimum concentrations of 100 μmol/l GP-2250 in SCC13cells and 50 μmol/l GP-2250 in A431 were observed to be the lowest dosescapable of significantly reducing cell viability and inhibitingproliferation rates in both cell lines. Comparing these dosages ofGP-2250 to the ones found in the study of Buchholz at al. (Innovativesubstance 2250 as a highly promising anti-neoplastic agent in malignantpancreatic carcinoma—in vitro and in vivo. BMC Cancer. 2017 Mar. 24;17(1)) with comparable assays, similar study design and analoguehandling of the assays (i.e. cell density, incubation time,measurements), it was found that the effect-related concentrations usedin this study are much lower. Buchholz at al. showed comparable effectsanalyzing the effect of substance GP-2250 on five different humanpancreatic cancer cell lines (AsPC-1, BxPC-3, MiaPaca-2, Panc-1,Panc-Tul) with a higher concentration range from 100 to 2000 μmol/l. Inthe Buchholz report, at a concentration of 1000 μmol/l more than 50%reduction of viable cells was monitored in four out of five cell lines.

The present disclosure also demonstrated was found that SCC13 cellsrequire higher concentrations of GP-2250 than A431 cells in order toachieve comparable antineoplastic, anti-proliferative and apoptoticeffects among the assays of the two cSCC cell lines (Table 2).

TABLE 2 Results of MTT and BrdU assays of GP-2250 in both cSCC celllines (SCC13 and A431) SCC13 A431 MTT cytotoxicity assay 42.74 (±1.43) 4.20 (±0.47) BrdU proliferation 62.40 (±1.11) 32.85 (±2.52) assay Table2 Results are represented as mean value in % (±SD in %) of viable cellsin the MTT assay and of proliferated cells in the BrdU assay forconcentrations of 200 μmol/l GP-2250

Analyzing both cell lines offers a possible explanation to thedivergence in cell responses, elucidating small differences betweentheir origins and characteristics. Both cell lines are human, epidermalsquamous carcinoma cells. While A431 cells are originally derived from a85-year-old female patient and characterised by p53-deficiency (23),SCC13 cells are derived from a primary facial cSCC tumor of a56-year-old female patient and are p53- also p16-deficient, probablyenhancing a quicker tumor cell replication, hence tumor proliferation.Differences in dose-related effectiveness between SCC13 and A431 mayfurther be due to inherent differences in cell turnover, unequalexpression of EGFR and sensitivity to reactive oxygen species (ROS).Excessive ROS production during mitochondrial oxidative metabolism canlead to oxidative stress and macromolecular damage in cancer.

Flow cytometry (FCM) analysis displayed in all cSCC cell lines adose-response correlation concerning relative distributions of viable,apoptotic and necrotic cells. Increasing concentrations of GP-2250caused a decrease of viable cells and an increase of apoptosis andnecrosis, with a predominant contribution of apoptosis to cell death.

Apoptosis leads to a caspase-dependent cell destruction with DNAfragmentation and cell shrinkage or to caspase-independent celldestruction with subsequent phagocytosis of the apoptotic cells.Considered the lack of phagocytosis in a cell culture setting due toabsence of inflammatory cells, a secondary necrosis follows, anautolytic process of cell disintegration, characterized by the sameappearance of primary necrosis. The necrosis marker (PI) used in thisstudy could ultimately not differentiate between a substance-inducedprimary necrosis or secondary necrosis—due to the lack of phagocytosis.The third type of programmed cell death, next to autophagy andapoptosis, which is amongst others activated through ROS, is programmednecrosis, causing cell swelling and membrane rupture.

ROS plays an important role in substance GP-2250 induced programmed celldeath (PCD). ROS expression under one and the same treatment differs inintensity in the same cancer type depending on whether tumor tissues areextracted from metastatic or non-metastatic cancer cells.

The compounds of the present disclosure are useful for inhibiting orreducing cancer cell migration. In some aspects, the subject has atumor, cancerous cells, pre-cancerous cells, or cancer stem cells, issuspected of having a tumor, cancerous cells, pre-cancerous cells, orcancer stem cells, or is at risk of developing a tumor, cancerous cells,pre-cancerous cells, cancer stem cells or metastases thereof, forexample, due to genetic, environmental and/or vocational factors. Insome aspects an effective dosage of the compounds of the presentdisclosure are administered to the subject to inhibit or reduce cancercell migration in the subject. As explained in Example 12, adose-dependent increase in inhibiting or reducing cancer cell migrationwas demonstrated.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the providedcomposition is mixed with at least one inert, pharmaceuticallyacceptable excipient and/or fillers or extenders (e.g., starches,lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g.,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents(e.g., agar, calcium carbonate, potato starch, tapioca starch, alginicacid, certain silicates, and sodium carbonate), solution retardingagents (e.g., paraffin), absorption accelerators (e.g., quaternaryammonium compounds), wetting agents (e.g., cetyl alcohol and glycerolmonostearate), absorbents (e.g., kaolin and bentonite clay), andlubricants (e.g., talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate), and mixtures thereof. Inthe case of capsules, tablets and pills, the dosage form may comprisebuffering agents.

The compounds of this disclosure, particularly compound 2250, have beenfound to be very soluble in water. In certain embodiments, no PVPnecessary to increase the solubility. For example, a 3.2% solution 2250is isotonic. This is an unexpected advantage over taurolidine.

Compounds of the invention, such as compound 2250 (with or withouttaurolidine and/or taurultam) are particularly useful in surgicaloncology, since the compounds do not hinder wound healing.Administration of other antineoplastic drugs must be delayed for up tofive weeks or more after surgery because other such antineoplastic drugshinder wound healing and promote anastomotic leakage. Such problems canbe avoided with compounds of the invention such as compound 2250, whichcan be administered during surgery and immediately thereafter, withoutwound healing issues or leakage issues.

Solid compositions of a similar type may be employed as fillers in softand/or hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the provided composition(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

In certain embodiments, capsules may contain an excipient formulationcontaining one or more of hydroxypropyl methylcellulose (HPMC), gelatin,and fish gelatin. In certain embodiments, a capsule may contain compound2250 in combination with taurolidine and/or taurultam. The capsule mayoptionally further contain one or more of lycopene, ellagic acid(polyphenol), curcumin, piperine, delphinidin, resveratrol,isothiocyanates such as sulforaphane, capsaicin, and piperlongumine.

Active compounds of the invention, such as compound 2250, can becombined with compounds such as gemcitabine. This combination can beused to treat cancers, such as pancreatic cancer. Taurolidine and/ortaurultam also can be combined with gemcitabine to treat, for example,pancreatic cancer.

In some embodiments, a nutritional cancer prophylaxis and treatmentproduct may contain 100-500 mg compound 2250 alone or in combinationwith 100-500 mg taurolidine and/or taurultam and one or more oflycopene, e.g., 20-200 mg, ellagic acid (polyphenol), curcumin, piperine(20-200 mg), delphinidin, resveratrol, isothiocyanates such assulforaphane, capsaicin, and piperlongumine.

It was unexpectedly found that the compounds could be administeredduring surgery and immediately after surgery because the compounds donot inhibit wound healing like other chemotherapy agents.

It was unexpectedly found that taurolidine, taurultam, andoxathiazin-like compounds and derivatives thereof kill tumor stem cells,which is very unusual and perhaps unknown among chemotherapy agents.Typical chemotherapy agents, if effective against tumor stem cells,generally are only effective at very high doses which are extremelytoxic to human patients.

It was unexpectedly found that lower doses of taurolidine and/ortaurultam killed tumor stem cells than were needed to kill tumor cells.

It was unexpectedly found that Oxathiazin-like compounds and derivativesthereof have a half-life in human blood that is significantly longerthan the half-life of taurolidine and taurultam. Accordingly, thesecompounds are cleared less rapidly from the bloodstream of the patients,thereby effectively delaying loss of drug potency caused by the body'sclearance mechanisms.

It was unexpectedly found that certain Oxathiazin-like compounds andderivatives thereof have reduced burning sensation when applied directlyinto tissue, unlike this effect observed in patients treated withtaurolidine.

It was unexpectedly found that the Oxathiazin-like compounds andderivatives thereof have a particularly advantageous combination ofproperties including high water solubility, versatile administrationroutes including oral and i.v., extended stability and half-life, andreduced side effect of burning sensation.

Thus, the half-life of compound 2250 is greater than 24 hours in humanblood, which is significantly higher than the half-life of taurolidine,which was found to be ˜30 minutes using the same test.

In one embodiment, the invention includes treating a patient byadministering compound 2250 to the patient that results in a baselineblood concentration of compound 2250 within about 5 minutes ofadministration. The method involves maintaining a blood concentration ofcompound 2250 in the patient that is about 80% of the baseline bloodconcentration for about 20 hours.

In one embodiment, the invention includes maintaining a bloodconcentration of an anti-neoplastic compound in a patient that is about80% of the patient's baseline blood concentration for about 20 hours byadministering a daily dosage of compound 2250 once daily to maintain theblood concentration that is 80% of the baseline blood concentration.

The daily dosage may be about 0.1 g to about 100 g, e.g., about 5 g toabout 30 g. The daily dosage may be administered in the form of anorally administrable composition. The daily dosage may be administeredin the form of a capsule, a tablet, or a pharmaceutically acceptablesolution. The daily dosage may be administered in a form that containscompound 2250 at a concentration of about 0.01 to about 3% w/v. Thedaily dosage may be administered in a form that contains compound 2250at a concentration of about 0.01 μg/ml to about 1000 μg/ml. The dailydosage may be administered in a form that contains one or moresolubilizing agents, e.g., polyols.

In some embodiments, the compounds are administered in compositions at aconcentration of about 0.01 to about 1000 μg/ml. In some embodiments,the compounds are administered in compositions at a concentration ofabout 1 to about 100 μg/ml. In some embodiments, the compounds areadministered in compositions at a concentration of about 10 to about 50μg/ml. The composition may also contain about 0.01 to about 1000 μg/ml,about 1 to about 100 μg/ml, or about 10 to about 50 μg/ml taurolidineand/or taurultam.

In some embodiments, the compounds are administered in compositions at aconcentration of about 0.01 to about 3%. In some embodiments, thecompounds are administered in compositions at a concentration of about0.1 to about 2.5%. In some embodiments, the compounds are administeredin compositions at a concentration of about 1% to about 2%. Thecomposition may additionally contain about 0.01 to about 3%, about 0.1to about 2.5%, or about 1 to about 2% taurolidine and/or taurultam.

In one embodiment, the oxathiazin-like compounds and derivatives thereofmay be administered as a co-therapy with taurolidine and/or taurultam tokill tumor stem cells. In accordance with such an embodiment, theco-therapy has been unexpectedly found to require a lower dosage of drugto kill tumor stem cells than necessary to kill normal tumor cells.

In certain embodiments, the oxathiazin-like compounds and derivativesthereof may be administered with Vitamin D3, which results to increasethe anti-tumor effects of the compounds.

In one embodiment, the compound is administered to the subject at atotal daily dose of from about 0.1 g to about 100 g, about 1 g to about80 g, about 2 g to about 50 g, or about 5 g to about 30 g.

Effective dosage amounts of the compounds are dosage units within therange of about 0.1-1,000 mg/kg, preferably 150-450 mg/kg per day, andmost preferably 300-450 mg/kg per day.

As used herein, the term pure refers to a substance that is at leastabout 80% pure of impurities and contaminants. In some embodiments, theterm pure refers to a substance that is at least about 90% pure ofimpurities and contaminants. In certain embodiments, the term purerefers to a substance that is at least about 95% pure of impurities andcontaminants. In some embodiments, the term pure refers to a substancethat is at least about 99% pure of impurities and contaminants. In someembodiments, the term pure refers to a substance that is at least about99.5% pure of impurities and contaminants.

In certain embodiments, compounds, compositions, and methods of thepresent invention encompass the use of micronized compounds. In someembodiments, the term “micronized” as used herein refers to a particlesize in the range of about 0.005 to 100 microns. In certain embodiments,the term “micronized” as used herein refers to a particle size in therange of about 0.5 to 50 microns. In certain embodiments, the term“micronized” as used herein refers to a particle size in the range ofabout 1 to 25 microns. For example, the size of the drug particles maybe about 1, 5, 10, 15, 20, or 25 microns.

In certain embodiments, compounds, compositions, and methods of thepresent invention encompass the use of nanoparticles. As used herein,the term “nanoparticle” refers to any particle having a diameter of lessthan 1000 nanometers (nm). In some embodiments, a nanoparticle has adiameter of less than 300 nm. In some embodiments, a nanoparticle has adiameter of less than 100 nm. In some embodiments, a nanoparticle has adiameter of less than 50 nm, e.g., between about 1 nm and 50 nm.Suitable formulations for injection or infusion may comprise an isotonicsolution containing one or more solubilizing agents, e.g., polyols suchas glucose, in order to provide solutions of increased compoundconcentration. Such solutions are described in EP 25366261. The solutioncan be rendered isotonic with ringer solution or ringer lactatesolution. The concentration of the compound in such solutions may be inthe range 1-60 g/liter.

In certain embodiments, exemplary compounds and processes for makingcompounds of the invention include the following:

The compounds may be in crystalline form, e.g., after crystallizationand/or recrystallization in an alcohol, ketone, ester, or combinationthereof. For example, the compounds of the present invention may becrystallized and/or recrystallized from an alcohol such as ethanol.

Exemplary compounds of the invention include the following:

It has been found that when used in the form of nanoparticles, thecompounds of the claimed invention achieve higher blood levels. In oneembodiment, the present invention includes compound 2250 alone or incombination with taurolidine and/or taurultam. For example, the presentinvention includes nanoparticles of the compounds of the presentinvention encapsulated in capsules.

In certain embodiments, the invention also relates to derivatives of theabove compounds having, e.g., activity as described herein of saidcompounds, for example, at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 100%, or more, of said activity.

In certain embodiments, the invention also relates to compositionscontaining the compounds described herein, including pharmaceuticallyacceptable solutions of said compounds, as well as orally administrablecompositions such as capsules and tablets containing said compositions.

In certain embodiments, the compounds of the present invention can beadministered to a subject or patient by any suitable means, for example,in solution, e.g., locally, systemically such as by intravenousinfusion, or the like.

Synthesis of 2250

-   -   sublimes in a vacuum at ˜70-80° C.

Starting Materials:

Isethionic Acid,

Carbylsulfat, Taurin, Taurinamide,

Cysteine, Isethionic Acid, inter alia

Synthesis 1 I.

Chemical synthesis

-   -   ethylenoxide with bisulfite

II. Isethionic Amide

Possible Alternative Chemical Synthesis Steps for 2250

Several Alternative Synthesis Steps for 2250 and 2255

I. Starting Materials 2250/2255

-   -   Synthesis sodium isethionate from Ethylenoxide+Sodium        hydrogensulfite        II. Reaction of Amine with Carbylsulfate

III.

Exemplary Synthetic Protocols I. Synthesis of 2244

2.15 g of pure 1907 was dissolved in 100 ml acetic acid ethyl ester, andcatalyzed using 0.5 g palladium on activated carbon. The solution washydrogenated at room temperature and atmospheric pressure. Thehydrogenation was complete after about 15 hours and the absorbed amountof hydrogen was 450 ml.

The hydrogenation was evacuated 3 times, each time with nitrogen, andthen the reaction mixture was filtered through a filter aid(diatomaceous earth). The clear colorless ethyl acetate solution wasconcentrated and dried in a rotary evaporator.

Yield: 1.25 g, which was innoculated with crystalized 2244.

Melting point: 42-44° C.

IR: corresponds to 2244, 99.3% pure.

II. Synthesis of 2244

5 g (0.023 mol) of 2264/1907 was boiled in 50 ml of concentrated HCl for3 hours under reflux, then allowed to cool to room temperature andseparated with 30 ml of dichloromethane in a separating funnel. Theaqueous phase was evaporated in a rotary evaporator and dried. A yellowoil remains which slowly crystallized after seeding with 2244 crystals.

IR corresponds to the substance 2244.

Recrystallized from ethyl acetate.

0.7 g obtained (24%).

Melting point: 44-45° C.

IR corresponds to the reference substance.

III. Synthesis of 2244

-   -   wherein Ph is a phenyl group.

230 mg 2269 was dissolved in 2 ml NaOH (1N) and refluxed at boiling witha reflux condenser for 15 minutes. The clear solution was cooled to 20°C. and acidified with hydrochloric acid. The resulting precipitate wasfiltered off under vacuum and dried.

Yield: 110 mg.

Melting point: 114-116° C.

IR showed 99% benzoic acid as by-product.

The acidic solution was concentrated to dry it on a rotary evaporatorand the solid was boiled with acetic ester. The ethyl acetate solutionwas filtered and concentrated to dryness under vacuum.

Weight: 110 mg. Oil was contaminated with oil and the IR peak for 2244(isethionic acid amide) was unclean.

The 110 mg was recrystallized from acetic ester.

Yield: 65 mg, Melting Point: 43-45° C.

IR corresponded to 52% 2244.

IV. Synthesis of 2244

-   -   wherein Ph is a phenyl group.

1.15 g 2269 was dissolved in 10 ml NaOH (1 N) and refluxed at boilingfor 15 minutes. The clear solution was cooled to 20° C. and acidifiedwith hydrochloric acid. The resulting precipitate was filtered off undervacuum and dried.

Yield: 0.5 mg.

Melting point: 114-116° C.

IR showed 82% benzoic acid by-product as control substance. Hydrolysisis not complete.

The acidic solution was concentrated to dry it on a rotary evaporatorand the solid was boiled with acetic ester. The ethyl acetate solutionwas filtered and concentrated to dryness under vacuum.

Weight: 0.8 g. Oil was contaminated with oil and the IR peak for 2244(isethionic acid amide) was unclean.

The 0.8 g was recrystallized from acetic ester.

Yield: 160 mg, Melting Point: 43-45° C.

IR corresponded to 26% 2244.

V. Synthesis of 2244

215 g 0.1 Mol 2264 and 1000 ml of concentrated hydrochloric acid (ca.36%) were boiled together for 30 minutes under reflux. The 2264 resolvedand there was an oily layer. The reaction mixture was allowed to cooland transferred to a separatory funnel where the oil was separated fromthe water phase. The acidic aqueous solution in which should be solvedisethionic acid amide (2244) was concentrated at 50° C. in a rotaryevaporator almost to dryness. The yellow oily residue was placedovernight in the refrigerator and 32.3 g of clear crystals were filteredoff under vacuum. Mp 43-45° C. IR: in oxygen having peaks at thefollowing wave numbers 655.82, 729.12, 844.85, 898.86, 947.08, 1003.02,1060.88, 1134.18, 1236.41, 1288.49, 1317.43, 1408.08, 1572.04, 3105.5,3209.66, 3313.82, and 3427.62 cm⁻¹ as shown in FIG. 7.

The mother liquor was concentrated to complete dryness.

VI. Synthesis of 2244

21.5 g 0.1 Mol 2264 and 100 ml of concentrated hydrochloric acid (ca.36%) were boiled together for 30 minutes under reflux. An oily layerformed and the reaction mixture was allowed to cool in a separatoryfunnel where the oil was separated from the water phase. The acidicaqueous solution in which the isethionic acid amide (2244) was dissolvedand shaken 2 times with methylene chloride, the methylene chloride wasseparated, and the acidic water solution was concentrated in a rotaryevaporator at 50° C. to dryness. The yellow oily residue was placedovernight in the refrigerator and 12.3 g of oil was obtained. Mp.:41-43° C. Analysis of the product showed that corresponds 99.8% to 2244by IR.

Distillation Experiment:

12.3 g were distilled under high vacuum:

Outside Temperature Inside Temperature Vacuum 190-210° C. 183-186° C.0.1 mm

Weight: 9.3 g of oil which was solid at room Temperature Mp: 43-45° C.

VII. Synthesis of 2244

2.0 g of pure compound 1907 was dissolved in 200 ml acetic ester and 0.5g palladium/activated carbon was added and the mixture was autoclaved at100° C. and hydrogenated at 50° C. After 6 hours run-time, the reactionmixture was allowed to cool overnight, and was then filtered andconcentrated to dryness under vacuum.

Wt.: 1.7 g oil—added CH2Cl2 and shaken, then allowed to stand—thensuction filtered result in crystalline solid having Wt.: 0.6 g, meltingpoint ca. 40° C.

For analysis 0.2 g of two-times acetic ester was added to crystallize.Melting point 43-44° C.

VIII. Synthesis of 2244

2.15 g of pure 1907 was dissolved in 100 ml acetic acid-ethyl ester,then added to 0.5 g palladium/activated carbon. Then the mixture washydrogenated at room temperature and atmospheric pressure. Hydrogenationwas terminated after approximately 15 hours. The absorbed amount ofhydrogen was approximately 450 ml. The hydrogen was then evacuated 3times and flushed with nitrogen, and then each reaction mixture wasfiltered through diatomaceous earth (celite). The clear, colorlesssolution, ethyl acetate was evaporated to dryness on a rotaryevaporator.

Wt.: 1.25 g oil which crystallized after seeding with 2244 crystals.

Melting point: 42-44° C.

IR: corresponds to 99.3% 2244.

IX. Synthesis of 2250

1.2 g pure 2245 pure was dissolved in 150 ml acetic acid purely solvedat 60° C. 0.3 g of palladium on activated carbon was added and wasstirred at 75° C. and the mixture was hydrogenated at atmosphericpressure.

Hydrogenation was stopped after 7 days. The absorbed amount of hydrogenwas approximately 480 ml.

The hydrogen was evacuated and purged 3 times with nitrogen.

Then the reaction mixture was filtered at 70° C. through a filter aid(Diatomaceous earth). The clear warm glacial acetic acid solution wascooled down to room temperature and white crystals were suctionfiltered.

Weight: 0.74 g, Melting Point: 225-227° C.

IR: 2245 corresponds to the starting material

The mother liquor was concentrated on a rotary evaporator to dryness.

Weight: 0.38 g of impure material was extracted with ethyl acetate.

The solution was concentrated.

Ethyl acetate Soluble Portion: Semi-solid substance obtained bysublimation;

Obtained 0.15 g semi-solid substance that was recrystallized from a fewdrops of water

Yield: 70 mg, Melting Point: 95-98° C.

IR corresponded to 98% 2250.

X. 1-Step Synthesis in High Yield of Sodium 2-BenzyletherEthanesulfonate

10.5 g sodium 2-bromoethanesulfonate was added to a solution of 110 mlbenzyl alcohol and 1.15 g sodium benzyloxide.

Then the mixture was boiled under reflux four times. The mixture wasthen concentrated under vacuum to dryness and then boiled with ethylalcohol three times. The alcohol was filtered and concentrated todryness.

The yield was 9.8 g and was confirmed by UV and IR.

Pure crystals were obtained by boiling the resultant sodium2-benzylether ethanesulfonate in ethyl alcohol, filtering, then coolingthe solution to crystallize pure sodium 2-benzylether ethanesulfonatecrystals out of solution.

XI. Synthesis of 2250

6.3 g vinylsulfonamide (from 2258),

50 ml of concentrated formic acid, and

1.1 g of paraformaldehyde were combined for 2 hours at reflux to producecompound 2250. Then, the clear acidic solution was concentrated on arotary evaporator to dryness.

Residue is: 5.9 g of pale yellow, honey-like syrup.

IR: Mixture of vinylsulfonamide and 2250

A 2 grams was sublimated and a few crystals were obtained.

Sublimate semisolid: IR: corresponds to 98% 2250.

XII. Synthesis of Vinylsulfonamide

Formyl isethionic chloride was placed in 50 ml of chloroform and wasplaced in a 350 ml sulfonation flask and cooled to −10° C. Then 25%ammonia gas was introduced. After introduction of the ammonia gas, theweight of the chloroform/NH3 was found to be 5 g. From −3° C. to 2° C.,the mixture was stirred slowly.

To 9.0 g distilled 2249

20 ml of chloroform was added drop wise. NH₄Cl precipitated immediately.

Then the ammonium chloride was filtered off under vacuum and the clearchloroform solution was concentrated in a rotary evaporator until dry.

Yield: 6.3 g of clear, thin oil.

IR: corresponds to 96% CH2=CH—SO2-NH2 (vinylsulfonamide).

XIII. Synthesis of 2261

300 g (1.26 mol) 2260 was weighed into a 750 ml multi-necked flask withKPG-stirrer.

415 ml trichloroethylene+phosphorus oxychloride (Density corresponds toabout 1.47 in 10% POCl₃) and

150 ml phosphorus oxychloride and 5.7 ml DMF was warmed to 105° C. whilestirring. The mixture was allowed to react for 5 hours.

The solid was filtered by vacuum and the liquid was distilled underwater-pump vacuum. The filter cake was washed with ethyl acetate. Afterdistilling off of trichloroethylene and phosphorus oxychloride, thewash-acetate was transferred into a flask and also distilled.

250 g (1.07 mol—85%) of a yellow liquid was collected. IR corresponds to2261.

XIV. Synthesis of 2250 and 2255:

XV. New Synthesis Schemes for Compound 2250 and Related Compounds:Starting Materials:

3-Hydroxypropane-1-sulfonic acid

3-Hydroxy-propane-sulfonic acid-γ-sultone (1,3-Propanesultone)

3-Hydroxy-propane-2-sulfonic acid

2-Hydroxy-propane-1-sulfonic acid

Compounds (Tetrahydro-Oxathiazine-Dioxide):

Chemical Intermediates

Protecting group: Benzyl chloride

Protecting group: Benzyl chloroformate

XVI. Synthesis of Precursor Compounds

Synthesis:

83.9 g vinylsulphonic acid sodium was added to a solution of 400 mlbenzylalcohol and 0.5 g sodium (catalytic amount) was added. The mixturewas warmed with stirring to 150° C. and most of the vinylsulphonic acidsodium went into solution. After 3 hours, the mixture was allowed tocool overnight and a thick solid crystallized. This solid wasvacuum-filtered and then suspended in ethyl alcohol, vacuum-filtered anddried.

Yield: 94.0 g, IR: corresponds to the desired compound (61.2% pure).

XVII. Synthesis of 1905

60 grams of vinylsulfonic acid sodium were added to a solution of 1000ml benzylalcohol and 0.5 g of sodium. Then, the whole mixture wasstirred under reflux and heated. After approximately 3 hours, the excessbenzyl alcohol was distilled and removed by vacuum and the rest wasboiled with alcohol. The alcohol solution was filtered, concentrated,crystallized to about ½,

37.3 g of a yellow cotton-wool-like substance was obtained.

The procedure was also repeated with 250 g vinylsulfonic acid sodium and2 liters of benzyl alcohol, processed as above and about 208 g wascrystallized.

The procedure was also repeated with 100 g vinylsulfonic acid sodium and1 liter of benzyl alcohol, processed as above and about 105 g wascrystallized.

The procedure was also repeated with 200 g vinylsulfonic acid sodium,processed as above and about 130 g was crystallized.

XVIII. Synthesis of 1906

6.7 g of 1905 (recrystallized) was added to 50 ml thionyl chloride and 1ml dimethyl formamide. The sodium salt dissolved immediately and themixture was heated to 40-50° C., let stand overnight at 20° C. andvacuumed until concentrated. Yield: 9.8 g, which was added to 50 ml NaOH2N and stirred well. The NaOH solution was washed with CHCl₃ and thenshaken with concentrated HCl to precipitate and captured with Na₂SO₄,then dried and distilled.

The process was repeated with 208 g 1905 mixed with 1000 ml thionylchloride and 10 ml dimethyl formamide. The mixture was refluxed and theexcessive thionyl chloride was distilled off until dry. The yield was250 g, which was processed as above.

XIX. Synthesis of 1907

9.8 g of 1906 was dissolved in chloroform (CHCl3) (turbid) andconcentrated into a portion of 150 ml concentrated ammonia in water andstirred. Stirring was continued for 3 hours with heating to 40-50° C.Then, the mixture was dried under vacuum and concentrated.

Yield: 3.1 g dark oil

The 3.1 g dark oil was added to 50 ml NaOH 2N and stirred well. The NaOHsolution was washed with CHCl₃ and then shaken with concentrated HCl toprecipitate and captured with Na₂SO₄, then dried and distilled. Yield:2.5 g oil

For analysis, a sample of 0.5 g was condensed at a temperature of 160°C., became solid and crystallized 3 times from ethyl acetate/benzene.

Melting point: 75-76° C.

Molecular formula: C₉H₁₃NO₃S

MW: 215.2

Calculated: C=50.23%, H=6.09%, N=6.51%, S=14.86%

Actual: C=50.14%, H=6.15%, N=6.35%, S=14.79%

XX. Synthesis of 1908

1.2 g of 1907 was dissolved in 200 ml ethyl acetate and 0.4 g Pdactivated carbon was added. The mixture was hydrogenated in ahydrogenated autoclave at 100 and at 50° C. for 4 hours. The mixture wasleft under pressure for a weekend at room temperature. Then the ethylacetate solution was filtered and dried under vacuum.

Yield: 1.1 g oil.

XXI. Synthesis of 1908

2 grams of 1907 were dissolved in 200 ml ethyl actetate and 0.5 gPd/Palladium/activated carbon was added. The mixture was hydrogenated ina high pressure autoclave at 100 and at 50° C. After 6 hours, thereaction mixture was left to cool overnight, then filtered and distilledunder vacuum until it dried to a residual oil.

Yield: 1.7 g oil.

CH2Cl2 was added, agitated and allowed to stand, crystallized, andseparated with suction under vacuum. Weight: 0.6 g, melting point about40° C.

Analysis:

0.2 g recrystallized 2 times from ethyl actetate.

Melting point: 43-44° C.

Molecular formula: C₂H₇NO₃S

MW: 125

Calculated: C=19.22%, H=5.65%, N=11.21%, S=25.65%

Actual: C=19.20%, H=5.67%, N=11.07%, S=25.73%

XXII. Synthesis of 1909

19.9 grams of 1906 were dissolved in 100 ml chloroform and added into asolution of 23 grams pure benzylamine and 200 ml pure chloroform.Immediately, benzylamine hydrochloride precipitated and the reactionmixture became warm. The mixture was then refluxed and the hydrochloridecompound was separated by suction and the clear CHCl3-mother liquor wasput into vacuum for drying.

Yield: 27 g yellow clear oil that slowly became solid.

The 27 g was dissolved into about 20 ml ethyl acetate and N-hexane(q.s.) was added so that the solution became nearly turbid. The mixturewas set aside in the cold overnight and it crystallized.

Yield: 9.2 g, melting point: 50-53° C.

For analysis, 1 g in N-hexane was recrystallized three times. Meltingpoint 56-57° C.

XXIII. Synthesis of 2260

0.675 mol of isethionic acid sodium salt (100.0 g) and 2.02 molbenzylchloride (233 mL) were mixed in a 750 mL multi-necked flask withKPG-stirrer. The mixture was heated at 70° C. inside temperature (95° C.outside temperature) and then Triethylamine (120 mL) was added drop wiseover one hour and the outside temperature was increased to 125° C. andmaintained. Subsequently, outside temperature increased to 140° C., andthe inside temperature rose to 130° C. A solid clustered at the stirrer,but went back into suspension. Hydrochloric acid vapors evolved.

30 mL of triethylamine was added drop wise and then reacted for 1.5 morehours. A viscous yellowish suspension formed. The product was allowed tocool to 50° C. inside temperature, then 300 mL water was added andvigorously stirred for 20 minutes and the mixture was transferred to a 2L separatory funnel. Then, the flask was rinsed out with 100 mL ofwater.

The combined aqueous phases were washed twice with 280 mLdichloromethane.

The aqueous phase was held at 40° C., while KCl was added to thesolution until saturated (about 130 g KCl). The mixture was filteredthrough a fluted filter and stored overnight in a refrigerator.

The remaining solid was extracted and dried, resulting in 30.85 g, yieldof 17.9%.

IR: OH band is present, similar to the precursor.

The mother liquor was again treated with KCl and stored (at 35-40° C.)overnight in the refrigerator.

Solid from the second precipitation with KCl was filtered off and dried,resulting in 60.0 g=34.9% and the IR corresponds to the desired product.

Solid 1: Was boiled with 150 mL EtOH and filtered while hot.

By repeated precipitating with KCl, boiling and crystallization, 32 g ofthe product were obtained for a yield of 19%.

XXIV. Synthesis of 2256

40 g taurinamide hydrochloride, 18 g Sodium nitrite and 300 ml ofdistilled water were boiled together under reflux until no more gas wascreated. The clear yellow solution was then cooled to 50° C.

30 ml of 1N NaOH was added to 10.5 g of acetaldehyde. The clear yellowsolution was left over the weekend under vacuum to dry. The result was arust-red honey-like residue weighing 37.6 g, which was extracted withethyl alcohol. The alcohol solution was filtered and concentrated on arotary evaporator to dry. The resulting dense oil residue was dissolvedwith ethyl acetate. The ethyl acetate solution was filtered, andconcentrated.

This resulted in 30.7 g of dense oil, rust-like color. From the denseoil, white crystals were isolated. The melting point is about 114-116°C.

The IR spectrum confirmed that the resulting compound had the structureof compound 2256:

In certain embodiments, a sublimation apparatus, comprised of laboratoryglassware known in the art, may be used in a technique of sublimation topurify compounds according to the invention. In certain embodiments, asublimation vessel is heated under vacuum and under reduced pressure.The compound volatizes and condenses as a purified compound on a cooledsurface, leaving non-volatile residue impurities behind. This cooledsurface often takes the form of a cold finger. After heating ceases andthe vacuum is released, the sublimed compound can be collected from thecooled surface.

In one embodiment, substituted derivatives compound 2250 may beprepared. Substituted derivatives of compound 2250 include:

Wherein R may be H or alkyl or aryl. In certain embodiments, R is a C₁to C₆ alkyl. In certain embodiments, R is methyl.

In certain embodiments, derivatives of compound 2250 are preparedaccording to the following reaction scheme:

In one embodiment, this disclosure includes a method of killing tumorstem cells by administering to a subject in need thereof a tumor stemcell killing effective amount of taurolidine, taurultam, or a mixturethereof. The tumor stem cell killing effective amount of taurolidineand/or taurultam is less than an amount of taurolidine and/or taurultamrequired for killing tumor cells.

In some embodiments, the taurolidine, taurultam, or a mixture thereof isadministered in a tumor stem cell killing composition at a concentrationof about 0.01 to about 500 μg/ml. In some embodiments, the taurolidine,taurultam, or a mixture thereof is administered in a tumor stem cellkilling composition at a concentration of about 0.1 to about 100 μg/ml.In some embodiments, the taurolidine, taurultam, or a mixture thereof isadministered in a tumor stem cell killing effective composition at aconcentration of about 10 to about 50 μg/ml. Taurolidine is effective atkilling tumor stem cells in tissue culture in vitro at 0.01 μg/ml.

In some embodiments, the taurolidine, taurultam, or a mixture thereof isadministered in a tumor stem cell killing composition at a concentrationof about 0.001 to about 2%. In some embodiments, the taurolidine,taurultam, or a mixture thereof is administered in a tumor stem cellkilling composition at a concentration of about 0.01 to about 1.5%. Insome embodiments, the taurolidine, taurultam, or a mixture thereof isadministered in a tumor stem cell killing composition at a concentrationof about 0.1% to about 1%.

In one embodiment, the taurolidine, taurultam, or a mixture thereof isadministered for tumor stem cell killing to a subject in need thereof ata total daily dose of from about 0.01 g to about 50 g, about 0.1 g toabout 30 g, about 0.5 g to about 10 g, or about 1 g to about 5 g.

Tumor stem cell killing effective dosage amounts of the taurolidine,taurultam, or a mixture thereof are dosage units within the range ofabout 0.01-500 mg/kg, preferably 1-100 mg/kg per day, and mostpreferably 5-50 mg/kg per day.

In another embodiment, this disclosure includes a method of killingtumor stem cells by administering to a subject in need thereof acompound selected from the following compounds:

wherein each R is independently H, alkyl, or aryl,

which may be used in combination with taurolidine and/or taurultam. Sucha technique provides a method for killing tumor stem cells using atleast two compounds having different half-lives, and thereby broadeningthe pharmacokinetic effects obtained thereby. In one embodiment,compound 2250 may be used in combination with taurolidine and/ortaurultam.

EXAMPLES Example 1

Anti-neoplastic activity of compound 2250

Introduction

Based on the recognition of taurolidine as a powerful anti-neoplasticagent, the analogue 2250 was synthesized by Geistlich Pharma.

Material and Methods

Chemicals: The compound 2250 and taurolidin 2% solution were provided byGeistlich Pharma AG, Wolhusen, assignee of the present invention.

Cell lines: The human glioma cell line LN-229 was used as describedpreviously (Rodak et al. 2005) as well as the human colon adenocarcinomacell line SW480.

Cytotoxicity assay: Dissociated LN-229 cells were seeded into 96-wellplates at a density of 10⁴ cells per well in 100 μl of culture medium.Approximately 24 h later, when the cells had reached 70-80% confluency,the medium was changed and treatment with compound #2250 (4.0-1000μg/ml), taurolidine (4.0-1000 μg/ml) or standard medium was started.Triplicate cultures were prepared for each sample. After 24 h ofincubation at 25° C., the remaining adherent viable cells were stainedusing crystal violet as described (Rodack et al. 2005). Cell viabilitywas determined by measuring the absorbancy at 540 nm. The results areexpressed as killing rate given by the difference between 100% of cellsand percentage of cells surviving. EC₅₀ values correspond to theconcentration inducing 50% cell death.

Results

Positive control: After incubating the human glioblastoma cells (LN-229)for 24 h with taurolidine, a concentration-dependent cytotoxicity wasdetermined (Tab. 1, FIG. 1) with an EC₅₀=45 μg/ml, a value whichcorresponds to earlier results obtained with this cell line (Rodack etal. 2005).

Test of 2250: When 2250 was incubated under the same experimentalconditions as taurolidine, a similar concentration-dependent loss ofcell viability was observed. The half-maximal concentration of inducingcell death was EC₅₀=50 μg/μl (Tab. 1, FIG. 1).

The results for SW480 cell cytotoxicities are shown in FIG. 2.

Discussion

The compound 2250 represents a new avenue in the search for novelantineoplastic agents of the taurolidine-type. Biologically, thecompound is as potent as taurolidine. Chemically, the compound showsstrikingly different features from taurolidine. By replacing a NH groupby an ether-oxygen, the double ring structure of taurolidine is avoided.Compound 2250 is a single ring structure and a close structural analogueof taurultam.

Mechanistically, the results show that the antineoplastic activity oftaurolidine is unlikely to be due to the formation of amethoxy-derivative, since 2250 is devoid of a methoxy group. Thecompound causes blebbing of tumor cells.

Summary

The compound 2250 shows potent antineoplastic activity in vitro, asdetermined for human glioblastoma cells (cell line LN-229). Its potency(EC₅₀=45 μg/ml) is comparable to that of taurolidine (EC₅₀=50 μg/ml) astested in the same cell line.

TABLE 1 Cytotoxicity of 2250 and taurolidine against LL-229 glioblastomacells. Concentration μg/ml 1000 500 250 125 62.5 31 15.5 8 4 —Taurolidine 0.109 ± 0.098 ± 0.165 ± 0.305 ± 0.317 ± 1.132 ± 1.434 ±1.478 ± 1.530 ± 1.435 ± OD ± SD 0.010 0.007 0.002 0.008 0.008 0.0420.031 0.040 0.026 0.009 Comp. 2250 0.189 ± 0.141 ± 0.120 ± 0.199 ± 0.372± 1.482 ± 1.482 ± 1.527 ± 1.477 ± 1.483 ± OD ± SD 0.007 0.007 0.0120.014 0.006 0.099 0.029 0.033 0.069 0.013The values were measured in triplicate and the OD is the absorbance at540 nm plus minus standard deviation (SD). High values correspond tohigh cell viability.

Example 2

The new compound 2250 (Tetrahydro1,4,5-oxathizain-4-dioxid) was testedand found to have a very high level of antibacterial activity againstStaphylococcus aureus and Escherichia coli. The antibacterial activityagainst Staph. aureus is about double as high as Taurultam.

Example 3

In punch plate tests, Compound 2250 was tested and found highly activeagainst MRSA lines 188, 189, 193, 194 and 195.

By displaying a combination of antimicrobial and antineoplasticactivity, compound 2250 is particularly suitable for surgical oncology.

Example 4

Each of compounds identified herein as compound 2250, 2255, 2245, A1,A3, B1, B2, or B3 is tested against cancer cell lines of cancersidentified herein, and found to be active against such cell lines.

Example 5

Each of compounds identified herein as compound 2250, 2255, 2245, A1,A3, B1, B2, or B3 is administered to patients having cancers identifiedherein, and is found to be effective in treating such cancers and safefor use in patients. Each of these compounds is administered withVitamin D3, a derivative, metabolite or analog thereof and thecombination is found to increase the anti-tumor effects of thecompounds.

Example 6

The half-life of compound 2250 in human fresh blood was measured at 37°C. in vitro by GC, PYE Unicam Series 204 FID.

Baseline Value: 49.0 ppm

After 1 hour: 50.6 ppmAfter 2 hours: 47.6 ppmAfter 20 hours: 38.6-39.0 ppm.

Thus, the half-life of compound 2250 is greater than 24 hours in humanblood, which is significantly higher than the half-life of taurolidine,which was found to be ˜30 minutes using the same test.

Example 7

Tissue samples from high grade gliomas WHO grade IV from newly diagnosedpatients (medium age of 54±10 years) were minced mechanically, digestedenzymatically and the dissociated cells were filtered. The isolatedtumor cells were cultured as bulk cells. Cancer Stem Cells (CSCs) wereisolated by the formation of neurospheres under neurosphere conditions(using neurobasal medium) from the murine SMA 560 glioma cell line orfrom freshly isolated human glioblastoma cells.

Cytotoxicity Assay

Bulk glioma tumor cells were cultured and incubated with taurolidine ortaurultam for 24 h or 48 h as described previously (Rodak et al., J.Neurosurg. 102, 1055-1068, 2005). CSCs were cultured for 7 days andsubsequently exposed to taurolidine, taurultam or temozolamide for 24hours. The number of remaining adherent cells were stained (crystalviolet or Alamar Blue) and quantified by absorbance measurements (540nm). Cell survival was expressed as the percentage of cells survivingrelative to the number of cells surviving in untreated control cultures.The results are given as % killing rate or EC₅₀ as the dose required forhalf-maximal cytotoxicity. Results

Cytotoxicity of Taurolidine and Taurultam Against Cancer Cells andCancer Stem Cells from the Mouse

The mouse SMA560 glioma cell line was used to provide tumor bulk cellsand CSCs. Following incubation of SMA560 bulk cells with variousconcentrations of taurolidine and taurultam (6.25, 12.5, 25, 50, 100,200 μg/ml), cytotoxicity was determined after 24 h and 48 h ofincubation. For both taurolidine and taurultam, a clear dose-dependentcytotoxicity was found with no major difference in potency between the24 h and 48 h time of incubation (FIG. 3A,B). The EC₅₀ value was 34.6μg/ml for taurolidine and 19.3 μg/ml for taurultam (FIG. 3C).

Mouse CSCs were generated from the SMA560 glioma cell line and culturedfor 7 days. The CSCs were treated with the same concentration oftaurolidine and taurultam as above and cytotoxicity was determined after24 hours. As shown in FIG. 4, both taurolidine and taurultam showed adose-dependent cytotoxicity with an EC₅₀ of 12.5 μg/ml for taurolidineand EC₅₀ of 10 μg/ml for taurultam against murine CSCs. These valuesdemonstrate for the first time that taurolidine and taurultam areeffective against a CSC.

Taurolidine and Taurultam Induce Cell Death in Human CSC Isolated fromFour Different Glioblastoma Patients.

CSCs were isolated from glioblastoma tissue resected from four patients.The same range of concentrations of taurolidine and taurultam wasapplied as above and the cytotoxicity was measured after 24 hours ofincubation with drug. All four glioblastoma CSCs tested (GBM #3, #4, #5and #6) were similarly sensitive to taurolidine and taurultam (FIG.5A,B). The mean EC₅₀ value of taurolidine was 13±2 μg/ml, the EC₅₀ valueof taurultam was 11±1.4 μg/ml (Table 2). In these experiments, thecytotoxic capacity of taurolidine and taurultam was compared with thatof temozolamide (TIM) applied in the concentration range of 5 μM to1,000 μM (FIG. 2C). The mean EC₅₀ value of TMZ was 68.5±26 μg/ml (Table2). Interestingly, this concentration is much higher than peak plasmalevels of TMZ measured in patients (13.7 μg/ml) (Portnow et al., ClinCancer Res 15, 7092-7098, 2009).

The results demonstrate that both taurolidine and taurultam areeffective against CSCs and this finding was established for glioma CSCsfrom two species, mouse and man.

The mouse CSCs were generated from a mouse glioma cell line (SMA 560).Remarkably, based on the EC₅₀ values, the CSCs were even more sensitiveto taurolidine and taurultam than the corresponding glioma bulk cells(about 3 fold for taurolidine and 2 fold for taurultam) (FIGS. 3, 4).

Human CSCs, freshly isolated from four human glioblastoma patients, werelikewise highly chemosensitive to both taurolidine and taurultam. TheEC₅₀ values for cytotoxicity were 13±2 ug/ml and 11±1.4 μg/ml,respectively (Table 2). These values demonstrate that the human CSCs,like their murine counterparts, are more sensitive to taurolidine andtaurultam (about 3 to 4 fold) than the human glioblastoma bulk cellswhich display EC₅₀ values in the range of 50 μg/ml (Rodak et al., J.Neurosurg., 102, 1055-68, 2005).

TABLE 2 Cytotoxicity Induced by taurolidine (Tau), taurultam (TT) ortemozolamide (TMZ) in cancer stem cells (CSC) derived from fourglioblastoma patients. EC₅₀ μg/ml = drug concentration resulting in 50%cell death compared to untreated control cultures in vitro. CytotoxicityCancer Stem EC50 (μg/ml) 24 h Cells n Taurolidine Taurultam TemozolamideGBM #3 3 15 10.5 84.4 (435 μM) GBM #4 2 12.5 12.5 97 (500 μM) GBM #5 214 11 48.5 (250 μM) GBM #6 3 10 9 44 (230 μM) Mean ± SD 13 ± 2 11 ± 1.468.5 ± 26

Example 8

Taurolidine and taurultam were tested against cancer stem cells derivedfrom a murine glioma cell line and human cancer stem cells. Taurolidineand taurultam were found to exert potent anti-neoplastic activityagainst cancer stem cells derived from a murine glioma cell line(EC₅₀=12.5 μg/ml for taurolidine, EC₅₀=10 μg/ml for taurultam) as wellas against human cancer stem cells, freshly isolated from fourglioblastoma patients (EC₅₀=13±2 μg/ml for taurolidine; EC₅₀=11±1.4μg/ml for taurultam).

Example 9 Antineoplastic Effect on Pancreatic Stem Cell-LikeMulticellular Spheroid Cultures.

Multicellular spheroids are composed of tumor cells growing in a3-dimensional structure stimulating the growth, micro-environmentalconditions and stem cell-like characteristics of real tumors. Themulticellular tumor spheroid (MCTS) model compensates for many of thedeficiencies seen in monolayer cultures. Spheroids on the scale of200-500 μm develop chemical gradients of oxygen, nutrients, andcatabolites, while having morphological and functional features similarto tumors. Therefore, assays utilizing the MCTS model allow for theassessment of drug penetration and are more predictive of in vivosuccess compared with monolayer cultures. MCTS assays are a tumor modelsystem of intermediate complexity between standard monolayer and tumorsin vivo.

Pancreatic tumor cells (Panc Tu-1, BxPC-3, Mia Paca-2, ASPC1) andpancreatic primary tumor cells (Bo80) were seeded in ultra-low adhesionplates in special stem cell media.

Pancreatic tumor cells (ASPC1, Mia Paca-2, Panc Tul, BxPC-3) andpancreatic primary tumor cells (Bo80) were raised in monolayer culturebefore seeding in ultra low adhesion plates under conditions of specialstem cell media to form multicellular spheroids and passed through acell strainer to exclude aggregates.

Half-maximal inhibition of cell viability was achieved with 750-1000 μMof compound 2250 in tumor cell lines AsPC-1, BxPC-3 and HCT-116. Theseeffects are similar to those observed in glioma cell line LN-229. Theinduction of cell death was due to apoptosis and necrosis (most likelynecroptosis). It was found that the induction of this programmed celldeath was prevented by addition of the reducing agent N-acetylcysteineand that caspases are not involved. Thus, there is a redox-directedmechanism of action.

The growth of pancreas tumor cells (AsPC-1, BxPC-3 and HCT-116) wasinhibited by compound 2250 with a half-maximal concentration of 300 μM,which is considerably lower than the concentration needed to elicitcytotoxicity.

As shown in FIG. 8, multicellular pancreatic tumor (Panc Tul or BxPC-3)spheroids were tested as control, taurolidine-treated (500 μM) orcompound 2250-treated (1000 μM) samples for 48 hours (columns labeledA). After treatment, each of the whole cell suspensions was passedthrough a 45 μm cell strainer again to analyze residual aggregates fortheir stability (columns labeled B).

FIGS. 9A and 9B show the results of FACS analysis of the Panc Tulmulticellular spheroid cultures CD133 content. CD133 is a known andwell-established hallmark of stem cells. The results show that theamount of CD133-positive cells in multicellular spheroid cultures ofPanc Tul was enriched 10-fold compared to Panc Tul grown in monolayerculture (B). Isotype IgG was used as negative control (A). The resultsdemonstrate that taurolidine and compound 2250 have an antineoplasticeffect on the pancreatic stem cell like multicellular spheroid cultures.

Example 10

In vivo study of taurolidine and compound 2250 as antineoplastic agentsin malignant pancreatic carcinoma.

The effects of taurolidine and compound 2250 were analyzed on nude mice(NMRI-Foxn1 nu/nu). 1×10⁷ tumor cells (PancTu-I and MiaPaca 2) wereinjected subcutaneously into the flank. The animals were randomized intothree groups: the control group; the group treated i.p. with taurolidine(TRD), and the group treated i.p. with compound 2250 (NDTRLT).

Tumors were grown to a size of 200 mm³ before the treatment was started.Mice were treated on alternating days with 500 mg/kg*body weight (BW).

As shown in FIG. 10A, administration of taurolidine decreased MiaPaca2tumor volume significantly compared to control (by about 2-fold).

As shown in FIG. 10B, administration of compound 2250 decreased MiaPaca2tumor volume significantly compared to control (by over 3-fold).

As shown in FIG. 10C, administration of taurolidine decreased PancTu Itumor volume significantly compared to control (by about 3-fold).

As shown in FIG. 10D, administration of compound 2250 decreased PancTu Itumor volume significantly compared to control (by about 2-fold).

The applied taurolidine and compound 2250 dosages showed no toxic effecton the mice during the study. In both tumor cell line models, asignificant reduction of tumor growth was obtained.

Tumor growth (volume) was significantly reduced from day 9 onwards(PancTuI) and day 11 onwards (MiaPaca2) versus controls. The dose of 500mg/kg i.p. was well tolerated with no overt sign of toxicity.

As shown in FIG. 11A, a xenograft model of pancreatic primary tumors (Bo73) was observed for 15 days and it was found that administration oftaurolidine slightly reduced relative tumor volume compared to controland administration of compound 2250 further reduced relative tumorvolume compared to control. However, the differences in tumor volumeswere not statistically relevant, likely due to the short duration of thestudy and the slow growth rate of the tumors. In FIG. 11B, a xenograftmodel of pancreatic primary tumors (Bo 70) was observed for 23 days andit was observed that administration of taurolidine and compound 2250significantly reduce tumor volume compared to control.

Administration, e.g., intraperitoneally, of taurolidine and/or compound2250 inhibits tumor growth in vivo.

Example 11 Cell Lines and Culture Methods

For all assays two cSCC cell lines were used, namely SCC13 and A431.SCC13 are human, epidermal, tumorigenic, p16- and p53-deficient squamouscarcinoma cells. A431 are epidermoid, tumorigenic, p53-deficientsquamous carcinoma cells, showing high EGFR expression.

The cSCC cell lines A431 and SCC13 cells were cultured in Dulbecco'sModified Eagle Medium (DMEM), supplemented with the antibioticspenicillin (100 U/ml), streptomycin (100 U/ml) and 2 mM L-Glutamine.Cells were grown in cell culture dishes (diameter 100 mm, surface 60cm²) in humidified 5% CO2 atmosphere at 37° C. and grown as monolayerfor at least 72 h.

Dose Finding Trials and Cell Characteristics

The different cell lines were incubated in a sub-confluent state withdifferent concentrations of the substance, compared to an untreatedcontrol for 6, 12, 24 and 48 hours, in order to determine thedose-effect-relation and to establish the most effective singleconcentration. Dose finding trials were performed using the MTTcytotoxicity assay, as described here below.

SCC13 are rapidly growing cSCC cells, which show a high proliferationrate. The most effective concentrations for all carried out SCC13 assayswere for substance GP-2250 increasing dosages of 100, 150, 200, 300, 400μmol/l. The A431 cells are slower proliferating cells than SCC13 cells.The most effective concentrations for all carried out A431 assays weretherefor increasing dosages of 50, 100, 125, 150, 200 μmol/l.

MTT Assay

In order to analyze and quantify the anti-neoplastic effect of substanceGP-2250 on cell viability and to determine its dose-response effect,colorimetric MTT assays were performed with both cell lines.

Cells were seeded to a density of 5×10³ cells/well in 96-well plates andincubated for 24 hours in order to obtain a sub-confluent monolayer. Themedium was then removed and cells were incubated for 6, 12, 24 and 48hours with new medium (100 μl/well) containing increasing concentrationsof substance GP-2250 and ddH2O as negative control. The most effectivedose-responses for SCC13 cells were 100, 150, 200, 300, 400 μmol/l ofsubstance GP-2250 and for A431 cells 50, 100, 125, 150, 200 μmol/l. Twohours before measurement, the yellow coloured MTT reagent3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (5 mg/ml)was administrated. It is reduced through mitochondrial dehydrogenase ofmetabolic viable cells into violet coloured formazan crystals, which canbe measured by photometry. The test media were removed and 100 μl DMSO(Dimethyl sulfoxide) was applied. After an incubation time of 10 min theviability of cells was analysed by using a microplate absorbance reader(ASYS, UVM340, Anthos Mikrosystheme GmbH, Germany), measuring theoptical density. The amount of violet formazan crystals is directlyproportional to the amount of viable cells. (25) For every cell line sixindependent assays were performed with consecutive cell passages.

BrdU Proliferation Assay, ELISA

In order to analyse and quantify the anti-proliferative effect ofsubstance GP-2250, BrdU (5-bromo-2-deoxyuridine) proliferation assays,ELISA (Roche Applied Science, Mannheim, Germany), were performed withall cell lines, according to the manufacturer's instructions (Version16, content version: January 2013, Cat. No. 11 647 229 001). Thisproliferation assay is a non-isotopic colorimetric immunoassay forquantification of BrdU incorporation into newly synthesised DNA ofactively proliferating cells.

Cells were seeded to a density of 8×10³ cells/well in 96-well plates andincubated for 24 hours in order to obtain a sub-confluent monolayer. Themedium was then removed and cells were incubated for 6 hours and 24hours with new medium (100 μl/well) containing increasing separateconcentrations of substance GP-2250 and ddH2O as negative control. Themost effective dose-responses for SCC13 cells were 100, 150, 200, 300,400 μmol/l and for A431 cells 50, 100, 125, 150, 200 μmol/l. After the6- or 24-hours incubation period the BrdU reagent was added foradditional 2 hours, before cells were introduced to the BrdUproliferation assay (Roche Applied Science, Mannheim, Germany), asdescribed by the manufacturer's instructions. BrdU is a nucleosideanalogue of (3H)-thymidine, which is incorporated into new strands ofsynthesised chromosomal DNA during the S-phase of the cell cycle. Theamount of cell proliferation was detected and measured via opticaldensity, using a microplate absorbance reader (ASYS, UVM340, AnthosMikrosystheme GmbH, Germany). All BrdU assays were performed with eightreplicates of three independent experiments with consecutive passages.The incubation time of 6 hours has been shown to be the most appropriatefor the BrdU proliferation assays.

Flow Cytometry (FCM)

Cells were seeded to a density of 2×10⁵ cells/well in 6-well plates andincubated for 24 hours in order to obtain a sub-confluent monolayer. Themedium was then removed and cells were incubated for 24 and 48 hourswith new medium containing increasing separate concentrations ofsubstance GP-2250 and ddH2O as negative control. The most effectivedose-responses for SCC13 cells were 100, 150, 200, 300, 400 μmol/l andfor A431 cells 50, 100, 125, 150, 200 μmol/l.

Cell numbers were determined individually from each dose-dependentsuspension, then fixed in 200 μl binding buffer (Bender MedSystems,Vienna, Austria) and 5 μl Annexin V-FITC (BD Biosciences, Heidelberg,Germany) was added. After 15 minutes incubation at room temperature andlight deprivation 10 μl Propidium iodide (PI) (Bender MedSystems,Vienna, Austria) was added. Cells were immediately analysed for AnnexinV-FITC and PI binding using a flow cytometer (FACS Calibur BDBiosciences, Heidelberg, Germany) and quantified through dot plotshistograms analysed by CellQuest Pro software (BD Biosciences,Heidelberg, Germany). Viability was defined by Annexin V-FITC and PInegative, apoptosis by Annexin V-FITC positive and PI negative andnecrosis by Annexin V-FITC negative and PI positive cells.

Statistical Analysis

The data of the anti-neoplastic effects of all MTT assays (percentage ofviable cells), BrdU proliferation assays (percentage of proliferatingcells) and FCM-analysis (percentage of viable, apoptotic and necroticcells) was statistically analysed with the data processing GraphPadPrism software (version 8.0). The results are expressed as mean value(%) and its standard deviation (±SD %). For the statistical comparisonbetween the experimental groups, considering normal distribution, aone-way ANOVA was performed, followed by a Turkey's post-hoc test.P-value less than 0.05 was considered statistically significant.Significance levels were categorised and indicated in the figures asfollows: *p≤0.05, **p≤0.01, ***p≤0.001 and n.s.=not significant.

Substance GP-2250 Showed a Significant Cytotoxic Effect on Both cSCCCell Lines

MTT assays were conducted with SCC13 and A431 cell lines in order toanalyse the effects of substance GP-2250 on cell viability. As indicatedin FIG. 12A-12B, both cell lines have been incubated for 24 hours withdifferent increasing concentrations of GP-2250: SCC13 cells wereseparately incubated with 100, 150, 200, 300 and 400 μmol/l; A431 cellswith 50, 100, 125, 150 and 200 μmol/l. Controls were set to 100% anddefined as baseline.

Compared to untreated controls (ddH2O), GP-2250 leads to a significantdose dependent reduction of cell viability, as revealed by opticaldensity measurement. The greater the concentration, the greater thecytotoxic effect of GP-2250 in both cell lines. Viability reduction of50% or greater compared to the untreated control (100%) was obtained forSCC13 cells with a concentration of 150 μmol/l GP-2250 showing 42.74%(±1.43%) cell viability and for A431 cells with a concentration of 100μmol/l showing 48.13% (±2.51%) (p<0.001) cell viability.

The maximum dose of GP-2250 led to an intense cytotoxic effect in allcell lines, leading with a concentration of 400 μmol/l in SCC13 cells toa viability of 9.09% (±0.85%) and respectively with a concentration of200 μmol/l in A431 cells to 4.20% (±0.47%) cell viability (Table 2).Even though concentrations were different in both cell lines, thedose-effect of each substance on viable cells were comparable andproportional in both MTT groups.

The IC₅₀ value was determined by the dose of GP-2250 which caused a 50%reduction in cell numbers compared to controls. The IC₅₀ for GP-2250varied between the two cell lines. IC₅₀ was obtained in SCC13 cells witha concentration of 190 μmol/l for substance GP-2250 and in A431 cellswith concentrations of 96 μmol/l respectively.

Substance GP-2250 Significantly Inhibited Proliferation in all cSCC CellLines

BrdU assays were conducted with both cSCC cell lines in order to analysethe effects of substance GP-2250 on cell proliferation. As depicted inFIG. 13A-13B, both cell lines have been incubated for 6 hours withdifferent increasing concentrations of GP-2250: SCC13 cells wereincubated with 100, 150, 200, 300 and 400 μmol/l; A431 cells with 50,100, 125, 150 and 200 μmol/l. Controls were set to 100% and defined asbaseline.

Compared to untreated controls (ddH2O), GP-2250 lead to a significantdose-dependent reduction of cell proliferation, as revealed by opticaldensity measurement. Lowest dosages of GP-2250 showed already asignificant reduction of proliferated cells compared to the untreatedcontrol (FIGS. 13A-13B). The greater the concentration, the fewer theamount of proliferating cells. The highest dosages of GP-2250 showed ahighly significant reduction of proliferated cells of more than 75%compared to the control in SCC13 cells, leading for a dosage of 400 μmolto 18.41% (±1.50%); a dosage of 200 μmol lead to 32.85% (±2.52) ofproliferation in A431 cells. The dose related effect of substanceGP-2250 can be characterized as comparable and proportional in both cSCCcell lines.

Substance GP-2250 Significantly Induces Apoptotic Cell Death in BothcSCC Cell Lines

FCM analysis was conducted to evaluate the impact of substance GP-2250on inducing apoptosis. SCC13 and A431 cells have been incubated for 24hours with substance GP-2250 in different concentrations, as summarizedin FIG. 14A-14F, and evaluated by FCM analysis, resulting in asignificant viable cell reduction and increase of apoptotic and necroticcells in comparison to controls (with ddH2O).

Compared to the respective negative controls, both cell lines showed asignificant total reduction of cell viability, being coherent to asignificant increase of apoptotic cells in all cell lines (FIG. 14A,14B, 14D, 14E). In SCC13 cells a significant reduction of cell viabilitywas observed at every dosage, reaching from 84.35% (±1.3%) at lowestdosage of 200 μmol/l GP-2250 to a cell viability of 52.89% (±2.9%) at adosage of 400 μmol/l GP-2250 (FIG. 14A).

In A431 cells a stronger reduction of cell viability was observed, withat highest dosage of 200 μmol/l GP-2250 a cell viability reduction to20.35% (±1.2%) (FIG. 14D). Similar significant cell line dependentpatterns were observed comparing the apoptotic and necrotic effect ofsubstance GP-2250, showing a greater contribution of apoptosis on celldeath than the one of necrosis in both cell lines (FIGS. 14B, 14C, 14E,14F). In the SCC13 cell line the highest used dosages of GP-2250 lead to38.15% (±1.3%) apoptotic cells and to 20.45% (±0.5%) of necrotic cells;in A431 cells 47.95% (±1.6%) and 36.46% (±1.6%) respectively (FIGS. 14B,14C, 14E, 14F).

Example 12

Cell motility of cSCC cells A431 and SCC13 was analyzed using cellmigration assays. Cells were seeded in 6-well culture plates to adensity of 6×10⁵ and grown to 100% confluence in approximately 24 hours,depending on the cell line. An artificial gap of approximately 1100 μmwide was then generated using a p200 pipet tip and cells were incubatedseparately with substance GP-2250 and ddH2O for negative control.Duplicate testing was performed for all concentrations. For each cellline the two closest concentrations to the IC₅₀ (half maximal inhibitoryconcentration) were chosen: 100 and 200 μmol/l GP-2250 for cell lineA431 and 100 and 150 μmol/l for the SCC13 cell line. Cells at the gapedge polarized and migrated into the gap space. The cell migration (gapclosure) was recorded using differential interference contrast (DIC)microscopy in order to not only observe and measure the cellprogression, but also to visualize the change in shape, size andbehavior of the incubated cells. Pictures were taken at 0, 6, 12 and 24hours as shown in FIGS. 15A and 15C.

Cell migration assays were conducted with SCC13 and A431 cell lines inorder to analyze and visualize the effects of substance GP-2250 on cellmigration. Both cell lines were separately incubated with 100 and 150μmol/l GP-2250 for the SCC13 cell line and 100 and 200 μmol/lrespectively for the A431 cell line. Duplicate testing was performed forall chosen concentrations. Cell migration was recorded using DICmicroscopy.

Compared to untreated controls, rising concentrations of GP-2250 lead tosignificant increasing anti-migratory effects (FIG. 15A-15D). While thegaps of the control groups measured after 24 hours 0.07% (±0.3%) in theSCC13 cells and in 6.36% (±0.3) in A431 cells, percentages of gapclosure were significantly higher with applied GP-2250 in both celllines leading to 47.26% (±0.8%) gap closure at highest dosages in SCC13cells and 73.03% (±1.0%) in A431 cells respectively.

Morphological changes in shape and size were observed after 24 hours ofincubation with substance GP-2250. Both cSCC cell lines exhibited on onehand cytoplasmic shrinkage and vacuolization and on the other handcytoplasmic swelling. Cells either detached from each other or floatedin the medium. These findings suggest substance induced apoptosis andrespectively necrosis.

Any of the above protocols or similar variants thereof can be describedin various documentation associated with a pharmaceutical product. Thisdocumentation can include, without limitation, protocols, statisticalanalysis plans, investigator brochures, clinical guidelines, medicationguides, risk evaluation and mediation programs, prescribing informationand other documentation that may be associated with a pharmaceuticalproduct. It is specifically contemplated that such documentation may bephysically packaged with an pharmaceutical product according to thepresent disclosure as a kit, as may be beneficial or as set forth byregulatory authorities.

While the subject matter of this disclosure has been described and shownin considerable detail with reference to certain illustrativeembodiments, including various combinations and sub-combinations offeatures, those skilled in the art will readily appreciate otherembodiments and variations and modifications thereof as encompassedwithin the scope of the present disclosure. Moreover, the descriptionsof such embodiments, combinations, and sub-combinations is not intendedto convey that the claimed subject matter requires features orcombinations of features other than those expressly recited in theclaims. Accordingly, the scope of this disclosure is intended to includeall modifications and variations encompassed within the spirit and scopeof the following appended claims.

1. A method of reducing or inhibiting cancer cell migration in a subjectin need thereof comprising administering an effective amount of acompound of formula I.

wherein R is H, an alkyl, or benzyl, to the subject
 2. The method ofclaim 1, wherein the compound is administered orally, intravenously,topically, or a combination thereof.
 3. The method of claim 1, whereinthe compound is


4. The method of claim 1, comprising administering 0.1 g to about 100 gof the compound per day.
 5. The method of claim 1, comprisingadministering 5 g to about 30 g of the compound per day.
 6. The methodof claim 1, comprising administering the compound in a gel, capsule,tablet, or solution.
 7. The method of claim 1, comprising administeringthe compound in a pharmaceutical composition at a concentration of about0.01 to about 3% w/v.
 8. The method of claim 1, comprising administeringthe compound in a pharmaceutical composition at a concentration of about0.01 μg/ml to about 1000 μg/ml.
 9. The method of claim 1, comprisingadministering the compound in a pharmaceutical composition containingabout 0.01 to about 3% taurolidine and/or taurultam.
 10. The method ofclaim 1, wherein the subject has a tumor, cancerous cells, pre-cancerouscells, or cancer stem cells, is suspected of having a tumor, cancerouscells, pre-cancerous cells, or cancer stem cells, or is at risk ofdeveloping a tumor, cancerous cells, pre-cancerous cells, cancer stemcells or metastases thereof.
 11. A method of treating a subject having asquamous cell carcinoma comprising administering an effective amount ofa compound of formula I.

wherein R is H, an alkyl, or benzyl, to the subject.
 12. The method ofclaim 11, wherein the squamous cell carcinoma is a cutaneous squamouscell carcinoma.
 13. The method of claim 11, wherein the compound isadministered orally, intravenously, topically, or a combination thereof.14. The method of claim 11, wherein the compound is


15. The method of claim 11, comprising administering 0.1 g to about 100g of the compound per day.
 16. The method of claim 11, comprisingadministering 5 g to about 30 g of the compound per day.
 17. The methodof claim 11, comprising administering the compound in a gel, capsule,tablet, or solution.
 18. The method of claim 11, comprisingadministering the compound in a pharmaceutical composition at aconcentration of about 0.01 to about 3% w/v.
 19. The method of claim 11,comprising administering the compound in a pharmaceutical composition ata concentration of about 0.01 μg/ml to about 1000 μg/ml.
 20. The methodof claim 11, comprising administering the compound in a pharmaceuticalcomposition containing about 0.01 to about 3% taurolidine and/ortaurultam.